New approach to linear gradient elution used for optimisation in reversed-phase liquid chromatography

A new mathematical treatment concerning the gradient elution in reversed-phase liquid chromatography when the volume fraction psi of an organic modifier in the water-organic mobile phase varies linearly with time is presented. The experimental ln k versus psi curve, where k is the retention factor under isocratic conditions in a binary mobile phase, is subdivided into a finite number of linear portions and the solute gradient retention time tR is calculated by means of an analytical expression arising from the fundamental equation of gradient elution. The validity of the proposed analytical expression and the methodology followed for the calculation of tR was tested using eight catechol-related solutes with mobile phases modified by methanol or acetonitrile. It was found that in all cases the accuracy of the predicted gradient retention times is very satisfactory because it is the same with the accuracy of the retention times predicted under isocratic conditions. Finally, the above method for estimating gradient retention times was used in an optimisation algorithm, which determines the best variation pattern of psi that leads to the optimum separation of a mixture of solutes at different values of the total elution time.     

High speed gradient elution reversed-phase liquid chromatography

A major disadvantage of gradient elution in terms of speed results from the need to adequately re-equilibrate the column. This work distinguishes two states of re-equilibration: (1) run-to-run repeatability and (2) full equilibration. We find that excellent repeatability (+/-0.002 min in retention time) is achieved with at most 2 column volumes of re-equilibration whereas full equilibration can require considerably more than 20 column volumes. We have investigated the effects of adding ancillary solvents (e.g. n-propanol, n-butanol) to the eluent and changing the particle pore size, initial eluent composition and type, column temperature and flow rate on the speed of full equilibration. Full equilibration seems to be more thermodynamically limited than kinetically controlled. Also, we show that the main limitation to reducing the full equilibration time is related to instrument design issues; a novel approach to overcome these instrumental issues is described.     

A new approach to the prediction of RP-HPLC retention times in gradient elution from isocratic data

Summary A general chromatographic model has been set up starting from a set of equations based on the concept of the velocity of a solute along the column. The composition of the mobile phase is taken into account solely as a numerical factor entering into suitable equations and totally independent of the chemical-properties of the constituents. A few isocratic experimental runs are necessary as input data, and subsequently a small amount of computational effort is sufficient to make predictions of retention times under gradient elution conditions for solutes of whatever chemical structure. The prediction errors are dependent on the steepness of the linear gradient chosen but are, in any case, acceptably low.     

Method to predict the bandwidth of elution profile under the linear gradient elution in reversed‐phase HPLC

Solute migration in a chromatographic column is an important consideration when designing batch or continuous chromatographic separation processes. Most design methods for the chromatographic processes are based on the equilibrium theory which concerns only the migration velocity of the solute. However, in real cases, it is important to predict the zone spreading which occurs by axial dispersion and mass transfer resistance. To predict the actual solute profiles in the column or effluent stream, numerical methods to solve nonlinear partial differential equations have been used. However, these methods involve much time and expense. In this work, two different rate factors are considered to predict the characteristics of the solute profiles. The first is solute migration velocity and the second is the zone spreading rate. The zone spreading rate can be estimated by the apparent axial dispersion coefficient which is obtained from the height of the equivalent theoretical plate in particular. Four benzene derivatives (benzene, toluene, p‐xylene, and acetophenone) were used as model solutes, and two mobile phase systems, water/methanol and water/ACN, were used in RP‐HPLC. The bandwidths and retention times of the solutes were predicted under several linear gradient conditions. The predicted and experimental bandwidths and retention times showed good agreement.     

Optimization of stepwise gradient elution in columns chromatography

Summary A general equation for the final retention of a solute chromatographed under conditions of stepwise gradient elution has been derived. The elution process and the distances travelled by solutes as a function of eluent volume were simulated by computer for the optimization of stepwise gradient prorams from isocratic HPLC data. The validity of the equations was experimentally veritied.     

Solvent gradient elution in micro HPLC

A simple gradient method for capillary column and micro high performance liquid chromatography using either single or multiple mixing chambers is presented. The gradient profile is dependent on the number of the mixing chambers. This system permits solvent gradient elution at flow rates less than 10 μl/min with satisfactory reproducibility.     

Methacrylate monolithic stationary phases for gradient elution separations in microfluidic devices

Methacrylate monolithic stationary phases were produced in fused-silica chips by UV initiation. Poly(butyl methacrylate-co-ethylene dimethacrylate) (BMA) and poly(lauryl methacrylate-co-ethylene dimethacrylate) (LMA) monoliths containing 30, 35 and 40% monomers were evaluated for the separation of peptides under gradient conditions. The peak capacity was used as an objective tool for the evaluation of the separation performance. LMA monoliths of the highest density gave the highest peak capacities (≈40) in gradients of 15 min and all LMA monoliths gave higher peak capacities than the BMA monoliths with the same percentage of monomers. Increasing the gradient duration to 30 min did not increase the peak capacity significantly. However, running fast (5 min) gradients provides moderate peak capacities (≈20) in a short time. Due to the system dead volume of 1 μL and the low bed volume of the chip, early eluting peptides migrated over a significant part of the column during the dwell time under isocratic conditions. It was shown that this could explain an increased band broadening on the monolithic stationary phase materials used. The effect is stronger with BMA monoliths, which partly explains the inferior performance of this material with respect to peak capacity. The configuration of the connections on the chip appeared to be critical when fast analyses were performed at pressures above 20 bar.     

Optimization of the mobile phase composition in gradient elution reversed-phase HPLC by stochastic prediction

Summary Quantitative analysis of more than ten compounds in a sample generally requires complex mobile phases to optimize the separation of the analytes by gradient elution reversed-phase HPLC. For this purpose, CHEOPS, a software package has been developed from the fully stochastic Computer Chromatogram Simulation Method. Calculation principles and optimization criteria are described. Experimental validation is presented with amino acids and steroids.     

Isocratic and gradient elution in micellar liquid chromatography with Brij‐35

Polyoxyethylene(23)lauryl ether (known as Brij‐35) is a nonionic surfactant, which has been considered as an alternative to the extensively used in micellar liquid chromatography anionic surfactant sodium lauryl (dodecyl) sulfate, for the analysis of drugs and other types of compounds. Brij‐35 is the most suitable nonionic surfactant for micellar liquid chromatography, owing to its commercial availability, low cost, low toxicity, high cloud temperature, and low background absorbance. However, it has had minor use. In this work, we gather and discuss some results obtained in our laboratory with several β‐blockers, sulfonamides, and flavonoids, concerning the use of Brij‐35 as mobile phase modifier in the isocratic and gradient modes. The chromatographic performance for purely micellar eluents (with only surfactant) and hybrid eluents (with surfactant and acetonitrile) is compared. Brij‐35 increases the polarity of the alkyl‐bonded stationary phase and its polyoxyethylene chain with the hydroxyl end group allows hydrogen‐bond interactions, especially for phenolic compounds. This offers the possibility of using aqueous solutions of Brij‐35 as mobile phases with sufficiently short retention times. The use of gradients of acetonitrile to keep the concentration of Brij‐35 constant is another interesting strategy that yields a significant reduction in the peak widths, which guarantee high resolution.     

Comparison of the sequential simplex method and linear solvent strength theory in HPLC gradient elution optimization of multicomponent flavonoid mixtures

Summary Using a camomile flavonoid extract as the sample and four different reversed-phase partition systems, the ability of the Simplex procedure to produce optimum gradient separation of unknown multicomponent mixtures was checked against the linear solvent strength (LSS) gradient elution theory. On the same partition systems the mean solvent strenghts were measured by experimentally determined logk vs. mobile phase composition plots. These mean solvent strengths are compared to those inferred from the optimum gradients and the usefulness of LSS theory in multicomponent mixture gradient elution optimization is discussed.Dadicated to Prof. Dr. A Liberti on the occasion of his 70th birthday.     

Separation of poly-3-hydroxyvalerate-co-3-hydroxybutyrate through gradient polymer elution chromatography

Polyhydroxyalkanoates are biodegradable polyesters produced by bacteria that can have a wide distribution in molecular weight, composition of monomers, and functionalities. This large distribution often leads to unpredictable physical properties making commercial applications challenging. To improve polymer homogeneity and obtain samples with a clear set of physical characteristics, poly-3-hydroxyvalerate-co-3-hydroxybutyrate copolymers were fractionated using gradient polymer elution chromatography (GPEC) as opposed to extensively used bulk fractionation. Separation was achieved using a reversed-phase column with chloroform and ethanol as the solvent and non-solvent, respectively. A separation was also conducted on a normal-phase column to compare elution patterns between columns of varied polarity. The fractions were analyzed using Size Exclusion Chromatography (SEC) and NMR to determine the percentage of 3-hydroxyvalerate in the copolymer as well as its molecular weight. It was found that as the percentage of "good" solvent was increased in the mobile phase, the polymers eluted with decreasing percentage of 3-hydroxyvalerate and increasing molecular weight which indicates the importance of precipitation/redissolution in the separation. The elution pattern of the polymer remained unchanged when using both a normal- and reversed-phase column which also illustrates the dominance of precipitation/redissolution in GPEC of polyhydroxyalkanoates. As such, GPEC is shown to be an excellent choice to provide polyhydroxyalkanoate samples with a narrower distribution in composition than the original bulk copolymer sample.     

Statistical scanning method for the optimization of gradient elution high performance liquid chromatography

Summary A computer-assisted method is presented for the optimization of separation in gradient elution reversed-phase HPLC. The method is based on a polynomial estimation from nine preliminary experiments according to a two-factor (initial solvent composition C and gradient time T) rectangular design. This is followed by a two-dimension computer scanning technique. Resolution is used as the selection criterion. Good agreement was obtained between predicted data and experimental results.     

DNA purification from crude samples for human identification using gradient elution isotachophoresis

Gradient elution isotachophoresis (GEITP) was demonstrated for DNA purification, concentration, and quantification from crude samples, represented here by soiled buccal swabs, with minimal sample preparation prior to human identification using STR analysis. During GEITP, an electric field applied across leading and trailing electrolyte solutions resulted in isotachophoretic focusing of DNA at the interface between these solutions, while a pressure‐driven counterflow controlled the movement of the interface from the sample reservoir into a microfluidic capillary. This counterflow also prevented particulates from fouling or clogging the capillary and reduced or eliminated contamination of the delivered DNA by PCR inhibitors. On‐line DNA quantification using laser‐induced fluorescence compared favorably with quantitative PCR measurements and potentially eliminates the need for quantitative PCR prior to STR analysis. GEITP promises to address the need for a rapid and robust method to deliver DNA from crude samples to aid the forensic community in human identification.     

Error analysis and performance of different retention models in the transference of data from/to isocratic/gradient elution

The transferability of retention data among isocratic and gradient RPLC elution modes is studied. For this purpose, 16 beta-blockers were chromatographed under both isocratic and gradient elution with acetonitrile-water mobile phases. Taking into account the elution mode where the experimental data come from, and the mode where the retention should be predicted, the following combinations are possible: isocratic predictions from (i) isocratic or (ii) gradient experimental designs; and gradient predictions from (iii) isocratic or (iv) gradient data. Each of these possibilities was checked using three retention models that relate the logarithm of the retention factor: (a) linearly and (b) quadratically with the volume fraction of organic solvent, and (c) linearly with a normalised mobile phase polarity parameter. The study was carried out under two different perspectives: a straightforward examination of the prediction errors and the analysis of the uncertainties derived from the variance-covariance matrix of the fitted models. The best combinations of prediction mode and model were: (i)-(b), (ii)-(c), (iii)-(b), and (iv)-(a) or (c).     

Modeling of salt and pH gradient elution in ion‐exchange chromatography

The separation of proteins by internally and externally generated pH gradients in chromatofocusing on ion‐exchange columns is a well‐established analytical method with a large number of applications. In this work, a stoichiometric displacement model was used to describe the retention behavior of lysozyme on SP Sepharose FF and a monoclonal antibody on Fractogel SO₃ (S) in linear salt and pH gradient elution. The pH dependence of the binding charge B in the linear gradient elution model is introduced using a protein net charge model, while the pH dependence of the equilibrium constant is based on a thermodynamic approach. The model parameter and pH dependences are calculated from linear salt gradient elutions at different pH values as well as from linear pH gradient elutions at different fixed salt concentrations. The application of the model for the well‐characterized protein lysozyme resulted in almost identical model parameters based on either linear salt or pH gradient elution data. For the antibody, only the approach based on linear pH gradients is feasible because of the limited pH range useful for salt gradient elution. The application of the model for the separation of an acid variant of the antibody from the major monomeric form is discussed.     

Application of artificial neural networks for gradient elution retention modelling in ion chromatography

Gradient elution in ion chromatography (IC) offers several advantages: total analysis time can be significantly reduced, overall resolution of a mixture can be increased, peak shape can be improved (less tailing) and effective sensitivity can be increased (because there is little variation in peak shape). More importantly, it provides the maximum resolution per time unit. The aim of this work was the development of a suitable artificial neural network (ANN) gradient elution retention model that can be used in a variety of applications for method development and retention modelling of inorganic anions in IC. Multilayer perceptron ANNs were used to model the retention behaviour of fluoride, chloride, nitrite, sulphate, bromide, nitrate and phosphate in relation to the starting time of gradient elution and the slope of the linear gradient elution curve. The advantage of the developed model is the application of an optimized two-phase training algorithm that enables the researcher to make use of the advantages of first- and second-order training algorithms in one training procedure. This results in better predictive ability, with less time required for the calculations. The number of hidden layer neurons and experimental data points used for the training set were optimized in terms of obtaining a precise and accurate retention model with respect to minimization of unnecessary experimentation and time needed for the calculation procedures. This study shows that developed, ANNs are the method of first choice for retention modelling of inorganic anions in IC.     

Computerized optimization of gradient elution conditions with ternary solvent mobile phases in RPLC

Summary In this work, an optimization procedure for gradient RPLC separation, using ternary mobile phases, is described. This procedure requires eight preliminary experiments in gradient elution mode to predict the retention surface for each solute over the whole triangular space. This is followed by computerized calculations to determine the best ternary gradient elution profile with respect to both selectivity and analysis time. The efficiency of this procedure from the point of view of rapidity and of accuracy, is illustrated for the specific separation of twelve phenyl urea herbicides.     

高效液相梯度洗脱法测定杏香兔耳风中绿原酸和芦丁

建立了反相高效液相色谱法同时测定不同来源的杏香兔耳风中的绿原酸和芦丁。采用Kromasil C18柱(4.6 mm i.d.×250 mm,5μm),以甲醇-0.5%H3PO4的水溶液为流动相,梯度洗脱程序为0 min(30∶70)-15 min(30∶70)-20min(50∶50)-40 min(50∶50);流速0.8 mL/min;检测波长350 nm;柱温25℃;光电二极管阵列检测器(PAD)。绿原酸线性范围为0.654~5.886μg,r=0.9997,样品的平均加标回收率为98.6%,RSD 1.7%;芦丁线性范围为0.218~1.962μg,r=0.9993,样品的平均加标回收率为97.1%,RSD 2.3%。本法可作为杏香兔耳风中绿原酸和芦丁的定量分析方法。